Purification of antibiotics with water insoluble carboxylic acids



Patented July 7, 1953 UNITED STATES PATENT OFFICE.

PURIFICATION OF ANTIBIOTICS WITH WATER INS OLUBLE CARBOXYLIC ACIDS NoDrawing. Application July 19, 1947, Serial No. 762,205

17 Claims.

This invention relates to basic antibiotics of the streptomycin type, i.'e.'., to members of the genus composed of streptomycin andantibiotically-active basic compounds which, like streptomycin, are.capable of forming water-soluble salts with acids such as sulfuric andwater-insoluble salts with organic-base-precipitating reagents (e. g.,derivatives of streptomycin, such as dihydrostreptomycin, andsimilarly-acting antibiotics, such as .streptothricin).

In 1944, Schatz, Bugie, and Waksman (Proc. Soc. Exp. Biol. Med. 1-944,57, 244) showed that a potent antibiotic, termed streptomycin, wasformed during the growth of the organism Actinomyces griseus (now calledStreptomyces griseus) and this antibiotic has since been demonstrated tobe of high clinical utility.

It was later found that a number of streptomycins are formed at the sametime. The first streptomycin obtained in pure crystalline form (as areineckate) and fully characterized (Wintersteiner and Friedapplication, Serial No. 666,541, filed May 1, 1946, :now Patent No.2,501,014, dated March 21, 1950-) is now referred to as streptomycin A;and the second streptomycin characterized (Fried and Titus application,Serial No. 737,400, filed March 26, 1947, now Patent No. 2,565,653,dated August 28, 195i) is now referred to as "streptomycin B. Moreover,there are indications that still other streptomycins are formed at thesame time and/or may be formed at the same time by changes in theculture conditions; and it is intended that each of such antibiotics andany mixtures thereof (whether in the form of the free base orWater-soluble salt thereof) be comprehended by the term streptomycinwhen employed unmodified hereinafter.

Streptomycin has been purified heretofore by various methods, all ofwhich were complex and/or ineflicient (and consequently expensive). Forexample, a method widely employed prior to this invention essentiallycomprised the following steps: (1) treating a primarystreptomycincontaining liquid with an activated charcoal, whichselectively adsorbs the streptomycin; (2) eluting the streptomycin fromthe charcoal with an aqueous, water-soluble mineral acid, preferably ata slightly elevated temperature (say about 3050 C.); (3) treating theeluate with an organic-base precipitating reagent, preferablyphosphotungstic acid; and (4) decomposing the precipitate. [The termprimary streptomycincontaining liquid comprehends, inter alia': (a) theculture liquid obtained by growing Strep tomyces griseus underconditions and in a {medium suitable for the production of streptomycinand removing the solids from the medium; ((1)) the culture liquid ofenhanced potency obtained by acidification of such culture (withhydrochloric or sulfuric acid, for example), neutralized; and (c) theliquid obtained by acidextraction of the solids separated from suchculture, neutralized] It is :the object of this invention to providesimple, efficient, and otherwise advantageous methods of purifying basicantibiotics of the streptomycin type-especially streptomycin; and it isa further object of this invention to provide certain salt-typederivatives of basic antibiotics of the streptomycin type useful inthese purification methods and for other purposes, and methods ofpreparing these derivatives.

The methods of this invention essentially comprise intimately contactingan aqueous solution of a basic antibiotic of the streptomycin type(especially streptomycin) with a substantially water-insolublecarboxylic acid (preferably, with a higher fatty acid, and notably withlauric acid) and a substantially water-immiscible organic solvent forthe carboxylic acid (preferably, with a substantially water-immisciblealiphatic alcohol, and notably with amyl alcohol mixtures); and thepurification methods of this invention essentially comprise intimatelycontacting an aqueous solution of an impure basic antibiotic of thestreptomycin type (c. g., a primary streptomycin-containing liquid, oran aqueous solution of a partially-purified streptomycin, such as theeluate referred .to hereinbefore) with a substantially water-insolublecarboxylic acid and a substantially water-immiscible organic solvent forthe carboxylic acid, recovering the organic solvent phase, andconverting the salt-type derivative of the antibiotic therein into awatersoluble salt of the antibiotic, preferably by intimately contactingthe organic solvent solution with an aqueous, water-soluble,relatively-strong acid (especially with an'aqueous, water-soluble,relatively-strong mineral acid), separating the aqueous phase, andrecovering the salt therein. The water-soluble salt of the antibioticthus -obtained is considerably purified, and the reacids utilizable inthe practice of this invention include, inter alia: aliphatic acids,such as lauric, Z-ethyl-hexoic, sorbic, mucic, caprylic, palmitic,stearic, and oleic acidsespecially coconut-oil fatty acids, palm-kernelfatty acids, or other technical lauric acids (1. e., mixtures of fattyacids having an average molecular weight approximating that of lauricacid) aromatic acids, such as benzoic, phenylacetic, p-amino-benzoic,3,5-dinitro-benzoic, cinnamic and a-dCCYl cinnamic; cycloaliphaticacids, such as camphoric and naphthenic; and heterocyclic acids, such asindoleacetic and furoic.

The substantially water-immiscible organic solvents for the carboxylicacid utilizable in the practice of this invention include, inter alia:aliphatic alcohols, such as n-amyl alcohol (l-pentanol), n-butanol(l-butanol), methyl isopropyl carbinol, methyl-isobutyl carbinol,isobutyl carbinol, 2-ethyl-hexanol, and amyl alcohol mixtures, such asrefined fermentationamyl-alcohol; aliphatic alcohol esters of lowerfatty acids, such as the acetate of methyl isobutyl carbinol; aliphaticketones, such as methyl isobutyl ketone; aliphatic ethers, such asdi-nbutyl ether and diethyl ether; hydrocarbons, such as benzene andtoluene, and halogenated hydrocarbons, such as chloroform and ethylenedichloride.

Among the water-soluble, relatively-strong acids utilizable for recoveryof the streptomycin from the organic solvent solution are sulfuric,hydrochloric, phosphoric, oxalic, citric, sulfamic, and nitric.

In one embodiment of the invention, a culture filtrate containing abasic antibiotic of the dried (i. e., frozen, and subjected to a highvacuum to sublime off the water). Alternatively, the intimate contactbetween the antibiotic solution, carboxylic acid, and organic solventmay be effected by first mixing the antibiotic solution with thecarboxylic acid (preferablyin aqueous suspension), and then intimatelycontacting the mixture with the organic solvent; or by first mixing theantibiotic solution with the organic solvent, and then intimatelycontacting the mixture with the carboxylic acid.

Preferably, especially when the aqueous solution of impure antibiotictreated is a culture filtrate, the organic solvent solution obtained iswashed with water before treatment with the aqueous acid, the washremoving a considerable amount of inactive solids but practically noneof the antibiotic.

The distribution coefficient of the antibiotic between the organicsolvent phase (solution of the carboxylic acid) and the aqueous phase isdependent upon pH. Thus, when using a refined fermentation amyl alcoholsolution of coconutoil fatty acids in the treatment of an aqueoussolution of streptomycin, maximum transference of streptomycin from theaqueous phase (as well as minimum transference of alkali soaps to theamyl alcohol phase) is obtained when the pH of the aqueous phase isadjusted to between about 6 and about 10, especially between about 6.5and about 8.5 (a transference of over 99% having thus been obtained froman aqueous solution of a streptomycin hydrochloride having a potency of422 units/mg. and contaminated with pyrogens, histamine-like substances,and streptidine). Other factors being equal, the pH should desirably behigher in the range specified as the purity of the antibiotic solutionis lower.

Where the carboxylic acid employed is in liquid form (as in the case ofZ-ethyl-hexoic or oleic acid), the organic solvent component may bedispensed with, an excess of the acid functioning as the organic solventcomponent. A separate organic solvent component may also be dispensedwith when using an excess of a low-melting carboxylic acid (orlow-melting mixture of carboxylic acids) and operating at a suitablyelevated temperature (high enough to liquefy the acid or mixture ofacids, but below that at which the antibiotic is destroyed).

Alternatively, the salt-type combination of the antibiotic and thecarbomlic acid may be recovered from its solution in the organic solventand then converted into a water-soluble salt, or used as a therapeuticagent per se. Thus, the salt-type derivative of the antibiotic may berecovered by evaporating off the organic solvent in vacuo', or (wherethe organic solvent lends itself to such procedure, as in the case ofbenzene, for example, by freeze-drying the solution). Alternatively, thesalt-type derivative of the antibiotic may be obtained more directly byinteracting a basic antibiotic of the streptomycin type with awater-soluble salt of a substantially waterinsoluble carboxylic acid, inan aqueous medium, as described and claimed in our application SerialNo. 762,206 filed of even date herewith (now Patent No. 2,631,143, datedMarch 10, 1953). These salt-type derivatives of antibiotics are, ingeneral, oil-soluble or oil-dispersible; and they may be usedtherapeutically, e. g., orally administered either per se or in oilymedia for the treatment of intestinal disorders (relying on intestinalprocesses to liberate the antibiotic in water-soluble form) orparenterally administered in oily media for prolonged antibiotic action.Where production of the salt-type derivative of the antibiotic-ratherthan purification of the antibioticis the objective, one may employ areconstituted aqueous solution of the antibiotic (e. g., an aqueoussolution of the highly purified or pure antibiotic).

The various extractions involved in the methods of this invention may ofcourse be carried out by the countercurrent technique; and the spentsolutions and/or extracts may be reemployed in the method for furtherremoval of activity and/or concentration of the extracted material.Thus, the spent antibiotic-containing liquid may be extracted with afresh batch of carboxylic acid and organic solvent; the spent organicsolvent may be extracted with a fresh batch of aqueous, water-soluble,relatively-strong acid, or such solvent may be used on a fresh batch ofantibioticcontaining liquid; and/or the aqueous acid extract may be usedto treat a fresh batch of organic solvent solution, to build up theantibiotic concentration therein.

The amount of carboxylic acid employed should desirably be sufficient tocombine with all of the antibiotic in the solution treated, andpreferably should be in excess.

The following examples are illustrative of the invention (all solutionsor dilutions referred to without identification of the solvent ordiluent being solutions in, or dilutions with, water) EXAMPLE 1 (a) 7.5liters of a streptomycin-containing culture filtrat (obtained, forexample, by growing Streptomyceus griseus in submerged culture in anaqueous medium containing soybean .meal, dextrose, and. sodium chloride,acidifying the in.- cubated culture, and filtering), having a potency of280 units/ml, is intimately contacted with a solution of (37.5 g.coconut-oil fatty acids in 750 ml. refined fermentation-amyl-alcohol.The pH of the aqueous phase is then adjustedto 6.9 by adding 25% NaOI-Isolution while stirring well (about ml. being required). The mixture isthen centrifuged, and the amyl alcohol layer formed (about 335 ml.) isrecovered; it has a streptomycin potency of about 1,520 units/ml, andhence contains about 25% of the activity-of the culture filtrate.

(b) 50 m1. of the amyl alcohol solution is intimately contacted with 50ml. Water; the pH of the aqueous phase is. adjusted to 3.4 by adding tothe mixture 10% hydrochloric acid (about 1.4, ml. being required); andthe aqueous layer (about 52 ml.) is separated. The amyl alcohol solutionis then intimately contacted with asccond 50ml. portion of Water, andthe pH of the aqueous phase adjusted to 2.85 by addition of 10%hydrochloric acid (about 0.7 ml. being required); and the aqueous layerformed (about 50 ml.) is separated. The aqueous layers are thencombined, neutral- (b) 1.4 liters of'thefir'st-obtained amyl alcoholsolution (potency 1,500 units/ml.)- is washed by intimately contactingit with 400 ml. distilled water andseparating the aqueous layer, thewash removing a considerable amount of inactive solids, but practicallyno streptomycin. The washed amyl alcohol solution is then intimatelycontacted with another 400 ml. portion of water, and '10 ml. normalhydrochloric acid is added (bringing the pH of the aqueous phase toabout 2.1) and theamyl alcohollayer formed on separation of the phasesis further extracted with a 200 m1. and then a 100 m1. portion of Water.The three aqueous extracts are combined, washed once by shaking with 100ml. ether, treated with l g. of a decolorizing carbon (e. .g. Darco(3-60), filtered, neutralized to pH 5.8 by addition of .normalsodiumhydroxide solution (about 14 ml. be ing required), and freeze-dried. Thestreptomycin hydrochloride thus obtained in a yield of about 5.48 g.has-a potency of about 4:94 units/mg. (the recovery from the amylalcohol solution being therefore practically quantitative).

For brevity, the details of other examples. procedurally analogous toExamples 1 and 2 but il lustrating variations of the invention are givenhereinafter in tabular form (Table 1), the organic-solvent solutionobtained being further treated as described herein-before, e. g insection b of Example 2, to obtain a. streptomycin hydrochloride.

Table I 'pH adjusted Approximate i li gziii hy iocl i l i id Organicsolvent used and Carboxylm acid'used (dis' (with'm percentage ExampleNo. containing Solution quantity solved in organic solsodiumhy.recoveryof treated, and potency vent) and quanmy tggi g tg s 1 liter(2.35 g. solids), refined fermentation amyl caprylic acid, 43.2 7 -1-57. o1, 95

422 units/mg. alcohol, 1 liter. 4 .do .d0 palmitic acid, 76.8g 7. 5 97 5lliter, 2,08011I11t5/ml. 2-ethrl-l1exo1cacid.65g 2, ethyl-.hexoic acid,66g" 6.3.5 75 o 10om1.,900units/ml refined fermentation amyl lauricacid, 622g 7.55 90 alcohol, 100 ml. 7,. 1001111., 940units/m1. n-butaol, 100 ml coconut oil't'atty acids, 6.8 85

' 0.22 g. 8., do methyl-isobutyl carbinol, do 7.4

100 ml. 9 do 2- ethyl-hexanol,100m1 d 7,7 7o 10 do di-n-buty'lether, 1001111;"; do 7.1 I 30 ized to pH 5.85 with 10% sodium hydroxide solu- XAM11 tion, and freeze-dried. The streptomycin hydrochloride thus obtainedin a yield of about 0.26 g,

has a potency of about 344 units/mg. (the recovery from the amyl alcoholsolution being therefore practically quantitative).

. EXAMPLE 2 (a) 14 liters of a streptomycin-containing culture filtrate(potency, 305 units/ml.) ,is intimately contacted with a solution of 103g. coconut-oil fatty acids in 2.2 liters refinedfermentation-amylalcohol. The pH of the aqueous phase is then adjustedto 7.20: 0.05 by adding 20% sodium hydroxide solution, While stirringwell (about 37 ml. being required); the mixture is centrifuged;

and the amyl alcohol layer formed (about 1,385

(a) 1 g. of an aqueous suspension of benzoic acid is added to 10 ml. ofa solution of partiallypurified streptomycin sulfate (potency, 6,550units/ml.), followed by 10 ml. ether; the pH is adjusted to 7-10 withdilute sodium hydroxide solution; the mixture is shaken for fiveminutes; and the ether layer formed is separated. About 40% .of theactivity is extracted into the ether layer.

procedure described in Example 1 or 2) to ob-' tain streptomycinhydrochloride (or other watersoluble salt of streptomycin).

EXAMPLE 12 (a) 24 g. of oleic acid is added to 2.65 liters of .astreptomycin-bydrochloride-containing eluate havinga potency of 556units/ml. (obtained by treating a primary streptomycin-containing liquidwith an activated charcoal, and elutlng the streptomycin from thecharcoal with dilute hydrochloric acid), followed by 530 ml. refinedfermentation-amyl-alcohol; the mixture is adjusted to pH 10 by additionof dilute sodium hydroxide solution, and stirred for 10 minutes; and theamyl alcohol layer formed on separation (containing about 90% of theactivity of the eluate) is recovered.

(b) The amyl alcohol solution is extracted with dilute sulfuric acid intwo fractions. First, the dilute sulfuric acid is added gradually whileagitating until the pH (originally about 9.6) reaches 6.5; the aqueouslayer formed (first extract) is separated; and a second extract isobtained by continuing the addition of dilute sulfuric acid until the pHreaches 1.9. On freeze-drying, the first extract yields about 4.5 g.streptomycin sulfate having a potency of about 163 units/mg.

(representing a recovery of about 55.3%); and

the second extract, on adjustment to pH 6.2 with dilute sodium hydroxidesolution and freeze-drying, yields about 1.1 g. streptomycin sulfatehaving a potency of about 321 units/mg. (and representing a recovery ofabout 25.8%, the total recovery being therefore about 81.1%.

EXANIPLE 13 '(a) One liter of refined fermentation-amylalcohol is addedto '7 liters of streptomycin-hydrochloride-containing eluate (potency,425 units/ml), followed by 54 g. of oleic acid; and the mixture isstirred while adding dilute sodium hydroxide solution until the pHreaches 10. The mixture is then stirred for an additional minutes; andthe amyl alcohol layer formed on separation (containing about 94.7% ofthe activity of the eluate) is recovered.

(b) The amyl alcohol solution is extracted 3 times by shaking 215 ml.portions of dilute sulfuric acid; and the three extracts are combined,neutralized with barium hydroxide solution, and filtered; and thefiltrate is freeze-dried. The streptomycin sulfate thus obtained has apotency of about 324 units/mg, the recovery from the amyl alcoholsolution being about 61.5%.

For brevity, the details of other examples procedurally analogous toExample 13 but illustrating further variations of the invention aregiven hereinafter in tabular form (Table II).

011 phase is adjusted to 6.7 by addition of dilute sodium hydroxidesolution; and the amyl alcohol layer formed on centrifugation (about2.5-liters, containing over about of the original activity) isrecovered.

(b) The amyl alcohol solution is washed four times, each time withone-fourth its volume distilled water (inorganic salts and organic basesweaker than streptomycin being removed by the washes, without materiallyreducing the streptomycin content of the amyl alcohol). The washed amylalcohol solution is then extracted with onefourth the volume ofthird-normal sulfuric acid, and then with two portions of distilledwater, each one-fourth the volume of the amyl alcohol solution; thethree extracts are combined and washed with one-fourth their combinedvolume of fresh amyl alcohol (to remove any entrained lauric acid); thewashed extract (whose pH is about 3.5) is separately treated with threeportions of an adsorbent carbon (e. g., Darco G-), 0.5 g. carbon permillion units streptomycin in the extract being used for each portion.The extract, which has thus been rendered practically water-white, isadjusted to pH 5.5-6.0 by addition of barium hydroxide solution; andafter standing several hours, the extract is filtered to removeprecipitated barium sulfate, partially concentrated at low temperaturein vacuo, and freeze-dried. The streptomycin sulfate thus obtained in ayield of about 12 g. has a potency of about 400 units/mg.

The following variations (either singly or severally) may be made in theforegoing example without material effect on recoveries: the lauric acidmay be replaced by a middle out of distilled coconut-oil fatty acids;the amount of acid used may be lowered to 200 g.; and the pH of theaqueous phase may be adjusted (by addition of a dilute solution of analkali) to any other other value within the range about 6.0 to about8.0. Also, the spent culture filtrate (after extraction of 50% of theactivity into the amyl alcohol phase) may be re-extracted with half thequantities of the amyl alcohol and lauric acid used for the firstextraction, about half the streptomycin remaining in the culturefiltrate being thus removed (and the extraction may be repeated as manytimes as economically feasible); and the Table II Data on productobtained by extraction of the organic solvent solution with dilutesulfuric acid, H d t d Approxitmatc neutralization. filtration p a 1115c percen age and freezoalrvin as Quantity of streptomy- Example cinhydrochloride- Organic solvent added, Carboxylic acid gi gag ssg figswuon (I!) of No. containing eluate and quantity added, and quantitydioxide S01u Organic p treated, and potency m Solvent solutionApproximate Approximate recovery potency, from organic units/mg. solventsolution 14 500 ml., 750 units/ml..." acetate of methyl isooleic acid,13.74 g 8.5 87 175 l butyl carbinol, 100 I m 500 ml., 625 units/inlbenzene, 100 ml oleic acid, 10.9 g 8. 5 96 210 500 ml., 414 units/rnlchloroform, 100 m1 "do 10 91.3 235 76 500 ml., 537 units/ml...n-butanol, 100 ml. 10 716 192 68 500 1111., 618 units/ml ethylenedichloride, 8. 5 97. 2 250 l 3 EXAMPLE 1 thus-extracted streptomycin maybe recovered (a) 25 liters of a streptomycin-containing culture filtrate(potency, 400 units/ml.) is mixed vwith v5 liters refinedfermentation-amyl-alcohol as streptomycin sulfate in the same manner asfrom the first extract, the product obtained being of substantially thesame quality as that obcontaining 600 g. lauric acid; the pl-I of theaque- 75 tained from the first xt t,

ing sodium hydroxide solution, whilej'stirring; the

mixture is stirred for an additional minutes; and the amyl alcohol phaseis recovered by centrifugation. (Re-extractions of the spent" culturefiltrate remove additional quantities of straptomycin.) I v Treatment ofthe amyl alcohol'solution as described hereinbefore to obtainstreptomycin [sulfate yields a product having a potency over about 400units/mg.

EXAMPLE 21 0.5 g. dihydrostreptomycin hydrochloride, having a potency of528 units/mg, is dissolved in 500 ml. water; the aqueous solutionis'mixed with a solution of g. coconut-oil fatty acids in 100 ml. of acommercial mixture of synthetic amyl alcohols (e. g., Pentasol); and thepH is adjusted to between 6.8 and 7.5 by addition of aqueous sodiumhydroxide solution in small increments, while stirring well. The amylalcohol layer, separated by centrifugation, contains over. about 80% ofthe activity of the aqueous solution treated. v The amyl; alcoholsolutionds then treated, as described hereinbefore, either to obtain thesalttype derivative of di-hydrostreptomycinper se, or to convert it intoa purified water-soluble salt of dihydrostreptomycin.

EXAIWPLE 22 21.2 mg. streptothricin hydrochloride, having a potency of500 units/mg. is dissolved in 21.2 ml. water; the aqueous solution ismixed with 12.7 ml. of a 20% solution of coconut-oil fatty acids in acommercial mixture of synthetic amyl alcohols (e. g., Pentasol) and themixture is adjusted to a pH of 6.5 to 7.1 by addition of normal sodiumhydroxide solution, while stirring. The separated amyl alcohol layercontains over about 87% of the activity of the aqueous solution treated.

Ihe amyl alcohol solution is then treated, as described hereinbefore,either to obtain the salttype derivative of streptothricin per se, or toconvert it into a purified water-soluble salt of streptothricin.

EXAMPLE 23 A streptomycin-containing culture filtrate having a potencyof 199 units/ml. ismixed with,4% of its volume of refinedfermentation-amyl-alcohol, and then extractedby a three-stagecountercurrent extraction with about one-seventh its volume of asolution of lauric acid in refined fermentation-amyl-alcohol, the ratioof lauric acid to culture filtrate being 8 g./liter, and the pH of theaqueousphase being adjusted continuously to 8.4 by addition of potassiumhydroxide solution. The amyl alcohol layer, separated by centrifugation,has a potency of about 1390 units/ml. (representing an extraction ofabout 93% of the activity of the culture filtrate) The amyl alcoholsolution is then treated, as described hereinbefore, either to obtainthe salttype derivative of streptomycin per se, or to convert it into apurified water-soluble salt of streptomycin.

Among other basic antibiotics of the streptomycin type utilizable forthe preparation of salttype combinations with substantially water-in- 10soluble carboxylic acids in accordance with this invention are pure (orsubstantially pure) strep.- tomycin A, streptomycin B;dihydrostreptomycin A, and dihydrostreptomycin B.

The basic antibiotic of the streptomycin type purified by the method ofthis invention may be further purified by repetition of the samepurification method. Also, it may. be preliminarily purified or furtherpurified by any other method,

especially by one of the following: (I) treating an aqueous solution ofthe antibiotic with a watersoluble salt of a substantiallywater-insoluble carboxylic acid, recovering the precipitated salttypecombination of the antibiotic and the carboxylic acid, and converting itinto a water.- soluble salt of the antibiotic (of. application SerialNo. 762,206 filed of even date herewith); (II) intimately contacting anaqueous solution of the antibiotic with a surface-active agent of theorganically-substituted polybasic-inorganic-acid type, recovering theprecipitated salt-type combination of the antibiotic and thesurface-active agent, and converting it into a water-soluble salt of theantibiotic (cf. application Serial No. 767,852 filed August 9, 1947, nowPatent No. 2,537,934, dated January 9, 1951; and (III) intimatelycontacting an aqueous solution of the antibiotic with .a surfaces-activeagent of the organically-substituted polybasic-inorganic-acid type and asubstantially water-immiscible organic solvent for soaps, recovering theorganic solvent phase, and converting the salt'ty pe derivative of theantibiotic therein into a water-soluble salt of the antibiotic (of.application Serial No. 767,851, filed August 9, 1947, now Patent No.2,537,933, dated January 9, 1951).

In the extraction of the organic solvent solution (of the salt-typcombination of antibiotic and carboxylic acid) with th aqueous,watersoluble, relatively-strong acid, a yellowish impurity is generallytransferred to the aqueous phase along with the streptomycin. When theextraction is modified, as illustrated by the following example, most ofthe yellowish impurity remains in the organic solvent phase: 1600 ml. ofan amyl alcohol solution of a salt-type combination of streptomycin andcoconut-oil fatty acids, obtained as described in section a, of Example2 and having a potency of 1175 units/ml, is extracted with threeseparate 400 ml. portions of 0.1 molar disodium phosphate solution,phosphoric acid being added to each extraction mixture until the pH ofthe aqueous phase is 6.60:0.02. After combining the three extracts andconcentrating to remove solvent, their total volume is about 950 ml.,and the potency is about1412 units/m1, indicating that about 70% of thestreptomycin activity of the organic solvent solution has beentransferred to the aqueous phase. The thus obtained aqueous streptomycinsolution containing bufie'r salts may be further purified by repetitionof the (carboxylic acid organic solvent) purification method of thisinvention.

The invention may be variously otherwise embodied within the SCOpe ofthe appended claims.

We claim:

1. The method which comprises intimately contacting an aqueous solutionof an antibiotic of the group consisting of streptomycin,dihydrostreptomycin, streptothricin, and water-soluble salts of thesewith a substantially waterinsoluble carboxylic acid and a substantiallywater-immiscible organic solvent for the carboxylic acid.

2. The method of purifying an antibiotic of the group consisting ofstreptomycin, dihydrostreptomycin, streptothricin, and water-solublesalts of these, which comprises intimately cont-acting an aqueoussolution of the impure antibiotic with a substantially water-insolublecarboxylic acid and a substantially water-immiscible organic solvent forthe carboxylic acid, recovering the organic solvent phase, andconverting the salt-type derivative of the antibiotic therein into awater-soluble salt of the antibiotic.

3. The method which comprises intimately contacting an aqueous solutionof a water-soluble salt of streptomycin with a higher fatty acid and asubstantially water-immiscible organic solvent for the acid.

4. The method which comprises intimately contacting an aqueous solutionof a water-soluble salt of streptomycin with lauric acid and asubstantially water-immiscible organic solvent for the acid.

5. The method which comprises intimately contacting an aqueous solutionof a watersoluble salt of streptomycin with a substantiallywater-insoluble carboxylic acid and a substantially water-immisciblealiphatic alcohol.

6. The method which comprises intimately contacting an aqueous solutionof a water-soluble salt of streptomycin with a substantiallywaterinsoluble carboxylic acid and refined fermentation-amyl-alcohol.

'7. The method of purifying streptomycin, which comprises intimatelycontacting a primary streptomycin-containing liquid with a substantiallywater-insoluble carboxylic acid and a substantially water-immiscibleorganic solvent for the carboxylic acid, recovering the organic solventphase, intimately contacting the organic solvent solution with anaqueous, water-soluble, relatively-strong acid, and recovering theaqueou ,phase.

8. The method of purifying streptomycin, which comprises treating aprimary streptomycin-containing liquid with an activated charcoal,eluting the streptomycin from the charcoal with an aqueous,water-soluble mineral acid, intimately contacting the eluate with asubstantially water-insoluble carboxylic acid and a substantiallywater-immiscible organic solvent for the carboxylic acid, recovering theorganic solvent phase, intimately contacting the organic solventsolution with an aqueous, water-soluble, relatively-strong acid, andrecovering the aqueous phase.

9. The method which comprises intimately contacting an aqueous solutionof a water-soluble salt of streptomycin with a solution of asubstantially water-insoluble carboxylic acid in a substantiallywater-immiscible organic solvent.

10. The method which comprises first mixing an aqueous solution of awater-soluble salt of streptomycin with an aqueous suspension of asubstantially water-insoluble oarboxylic acid, and then intimatelycontacting the mixture with a substantially water-immiscible organicsolvent for the carboxylic acid.

11. The method which comprises first mixin an aqueous solution of awater-soluble salt of streptomycin with a substantially water-immiscibleorganic solvent for substantially waterimmiscible carboxylic acids, andthen intimately contacting the mixture with a substantiallywater-immiscible carboxylic acid.

12. The method of purifying streptomycin, which comprises intimatelycontacting an aqueous solution of an impure water-soluble salt ofstreptomycin with a substantially water-insoluble carboxylic acid and asubstantially waterimmiscible organic solvent for the carboxylic acid,the pH of the aqueous phase being adjusted to between about 6 and about10, recovering the organic solvent phase, intimately contacting theorganic solvent solution with an aqueous, watersoluble,relatively-strong acid, and recovering the aqueous phase.

13. The method which comprises intimately contacting an aqueous solutionof Water-Soluble salt of streptomycin with an excess of a substantiallywater-insoluble carboxylic acid in liquid form.

14. The method which comprises intimately contacting an aqueous solutionof a Water-soluble salt of streptomycin with an excess of 2-ethyl-hexoic acid.

15. The method which comprises intimately contacting an aqueous solutionof an antibiotic of the group consisting of streptomycin,dihydrostreptomycin, streptothricin, and water-soluble salts of thesewith a substantially waterinsoluble carboxylic acid and a substantiallywater-immiscible organic solvent for the car boxylic acid, separatingthe organic solvent phase, and recovering the salt-type combination ofthe antibiotic and the carboxylic acid from its solution in the organicsolvent.

16. The method which comprises intimately contacting an aqueous solutionof a member of the group consisting of streptomycin and a water-solublesalt thereof with a substantially Water-insoluble fatty acid and asubstantially water-immiscible alcoholic solvent containing from 4 to 6carbon atoms.

1'7. The method of purifying streptomycin which comprises intimatelycontacting an aqueout solution of an impure member of the groupconsisting of streptomycin and water-soluble salts thereof with asubstantially water-insoluble fatty acid and a water-immisciblealcoholic solvent containing from 4 to 6 carbon atoms, recovering thealcoholic solvent phase, and converting the salt-type derivative ofstreptomycin therein into a water-soluble salt of streptomycin.

WILLIAM A. LOTT.

WILLIAM BRAKER. ANDREW ELLIS OKEEFFE.

References Cited in the file of this patent UNITED STATES PATENTS NumberName Date 2,139,839 Mckinney Dec. 13, 1938 2,481,267 Walti Sept. 6, 19492,501,014 Wintersteiner et al. Mar. 21, 1950 OTHER REFERENCES McElvain,Characterization of Organic Compounds, 1945, page 64, 1 page.

Fried et al., Science, v. 101 (1945), pages 613-615, 3 pages.

Kuehl et al., Science, v. 102 (1945), pages 34-35, 2 pages.

Kolmer et al., Science, v. 104 (1946), pages 315-317, 3 pages.

1. THE METHOD WHICH COMPRISES INTIMATELY CONTACTING AN AQUEOUS SOLUTIONOF AN ANTIBIOTIC OF THE GROUP CONSISTING OF STREPTOMYCIN,DIHYDROSTREPTOMYCIN, STREPTOTHRICIN, AND WATER-SOLUBLE SALTS OF THESEWITH A SUBSTANTIALLY WATERINSOLUBLE CARBOXYLIC ACID AND A SUBSTANTIALLYWATER-IMMISCIBLE ORGANIC SOLVENT FOR THE CARBOXYLIC ACID.
 8. THE METHODOF PURIFYING STREPTOMYCIN, WHICH COMPRISES TREATING A PRIMARYSTREPTOMYCIN-CONTAINING LIQUID WITH AN ACTIVATED CHARCOAL, ELUTING THESTREPTOMYCIN FROM THE CHARCOAL WITH AN AQUEOUS, WATER-SOLUBLE MINERALACID, INTIMATELY CONTACTING THE ELUATE WITH A SUBSTANTIALLYWATER-INSOLUBLE CARBOXYLIC ACID AND A SUBSTANTIALLY WATER-IMMISCIBLEORGANIC SOLVENT FOR THE CARBOXYLIC ACID, RECOVERING THE ORGANIC SOLVENTPHASE, INTIMATELY CONTACTING THE ORGANIC SOLVENT SOLUTION WITH ANAQUEOUS, WATER-SOLUBLE, RELATIVELY-STRONG ACID, AND RECOVERING THEAQUEOUS PHASE.